Method of Forming Non-Immunogenic Hydrophobic Protein Nanoparticles, and Uses Therefor

ABSTRACT

Methods are described for producing non-immunogenic nanoparticles from protein sources by controlling the pH in a nanoprecipitation process. The nanoparticles that are produced by the disclosed methods range in diameter size from about 100 ran to about 400 nm, with a preferred diameter size of from approximately 100 nm to approximately 300 nm, thereby rendering them non-immunogenic. The invention further discloses methods for producing nanoconjugates that are suitable for a variety of therapeutic, diagnostic and other uses.

TECHNICAL FIELD

This invention relates to methods of forming nanoparticles, andspecifically relates to methods of forming nanoparticles fromhydrophobic, water-insoluble protein-based polymers to producenon-immunogenic delivery systems for use in pharmaceutical, therapeuticand diagnostic applications.

BACKGROUND

The references discussed herein are provided solely for the purpose ofdescribing the field relating to the invention. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate a disclosure by virtue of prior invention.

Zein is a plant protein isolated from corn or maize and belongs to afamily of prolamines which are composed of high amounts of hydrophobicamino acids, such as proline, glutamine and asparagine. Zein is clear,odorless, non-toxic, biodegradable and water-insoluble. Zein has beeninvestigated and used as a polymer in the pharmaceutical, medical, food,cosmetic, adhesive and packaging industries.

In the food and pharmaceutical industries, zein has been used, forexample, to film-coat materials and to form particulate systems such asmicroparticles or nanoparticles [1-5]. Various methods of forming zeinparticles have been proposed. For example, U.S. Pat. No. 5,330,778, thecontents of which are incorporated herein, discusses a method forpreparing microparticles using zein, and uses pH alteration to form thezein microparticles[6]. However, the method described in U.S. Pat. No.5,330,778 produces zein particles with larger micron sizes and with awide particle size distribution, which has significant drawbacks, forexample, for in vivo use.

It is important to ensure that a biomaterial used for human or animalapplications is safe and non-immunogenic. In general, upon in vivoadministration (e.g., introduction into the body) of particles,phagocytic cells in the blood and tissues, which are responsible forimmunological recognition and removal of foreign particles, can initiatean immune response depending on the physicochemical characteristics ofthe particles. The uptake by phagocytic cells is dependent on bothparticle size and surface hydrophobicity of the foreign particle. Ingeneral, particles in a diameter size range greater than approximately500 nm are prone to phagocytosis. Particles with a hydrophobic surfaceare easily recognized by the phagocytic cells [7]. For example, Lopezand Murdan [8] have recently reported that zein microspheres of adiameter of 1.36±0.036 are immunogenic and, consequently, are notsuitable as a drug, vaccine or other therapeutic carrier.

SUMMARY OF THE INVENTION

In one aspect of the disclosure, the present invention generally relatesto a method for producing very small particles, or nanoparticles. Theparticles may be formed from hydrophobic water-insoluble proteinsincluding, for example, zein.

In another aspect of the disclosure, methods are employed to producenanoparticles that reduce or substantially overcome the immunogenicitythat is experienced in the use of larger-sized nanoparticles ormicroparticles, including those formed from, for example, hydrophobicwater-insoluble proteins. The non-immunogenic effect of thenanoparticles made in accordance with the methods of the presentinvention is achieved by controlling the size of the particles formed bythe method, as well as the range of particle sizes.

In some implementations of the invention, the range of particle diametersizes is less than approximately 400 nm. In preferred implementations ofthe invention, the range of particle diameter sizes is less thanapproximately 300 nm, and in some further implementations the range ofparticle diameter sizes is approximately 100 nm to approximately 300 nm.While size is discussed in this disclosure in terms of a diameter, thisshould not be interpreted to imply that the nanoparticles discussedherein are perfectly spherical in shape, although spherical shapes inthe nanoparticles may be achieved. It should be understood that thedimensions disclosed herein may simply be measured between oppositesides of the particle, or the largest dimension across the particle fromopposite sides.

In one aspect of the invention, the methods of the invention may becarried out using water-insoluble hydrophobic proteins that may bederived from a variety of sources including plant, animal and syntheticsources. In various aspects, the method may be carried out with a familyof prolamines which are composed of high amounts of hydrophobic aminoacids such as, for example, proline, glutamine and asparagine. Thesehydrophobic amino acids make the protein water-insoluble. The prolaminesmay be found in various grains such as corn, wheat, barley, rice,sorghum, and in other plants and animal sources. Some examples ofsuitable prolamines are zein, gliadin, hordein and kafirin, although theapplication of the method is not necessarily limited to these examples.For the purposes of this description, and merely as one exemplarillustration of the invention, the methods are described herein usingzein, by way of example only.

In various implementations of the method, white zein is utilized toproduce nanoparticles in a desirable diameter size range ofapproximately 100 to approximately 400 nm. It has been found that theuse of yellow zein may produce particles with relatively larger diametersize, and may also produce particles with wider particle diameter sizedistribution. It is believed that the pigments in yellow zein may affectthe solubility of the yellow zein and the nanoparticle formation usingyellow zein.

The methods of the invention produce nanoparticles of a generallysmaller diameter size and narrower diameter size range than wouldotherwise be possible. These smaller nanoparticles are achieved byimplementing a pH-controlled nanoprecipitation process using one or moreparticular grades of a base protein, such as zein, and by using variouscombinations of buffers, surfactants, and phospholipids that areselected to achieve nanoparticle sizes and diameters that render thenanoparticles non-immunogenic.

The methods of the disclosure are further suitable for preparingnanoparticles with a wide variety of molecules, particles or agents,having varying physicochemical properties, to form encapsulated,absorbed, complexed or conjugated materials with the nanoparticles. Forexample, the method may be utilized to entrap small hydrophilicmolecules, small hydrophobic molecules and macromolecules. In each ofthese examples, an encapsulation efficiency of approximately 60% toapproximately 80% may be achieved. The nanoparticles formed inaccordance with the present invention may be able to provide sustaineddelivery of the encapsulated molecule for up to a week, or possiblymore, in an in vitro and in vivo environment.

In one aspect of the invention, methods are employed to producetherapeutic and/or diagnostic nanoparticles, e.g., an anticanceragent-containing nanoparticles. Such nanoparticles can provide targeteddelivery and temporal control of the release of an active agent, whichis often a therapeutic agent such as a small molecular drug, nucleicacids, protein, vaccine, antibody, chemical or other agent or substance.In addition to the therapeutic methods described, the invention providesmeans for producing nanoparticles with diagnostic moieties, e.g.,imaging agents, probes, and the like.

In a further aspect of the invention, a kit is provided for preparationof nanoparticles in accordance with the methods of the invention. Thekit contains a selected amount of a water-soluble protein, at least onebuffering agent and at least one surfactant. The kit may also include ahydroalcoholic solvent. The kit may also include at least onephospholipid the amount of which may be selected to provide a selectedratio of phospholipids to surfactant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates by means of a flow chart the general steps of formingblank zein nanoparticles in accordance with the method of the presentinvention.

FIG. 2 illustrates by means of a flow chart the steps of forming 6,7hydroxy coumarin-loaded nanoparticles in accordance with the invention.

FIG. 3 depicts various electron microscopy microphotographs of zeinnanoparticles. FIG. 3( a) is a scanning electron microphotograph ofblank zein nanoparticles. The particles are shown to be spherical andwith a smooth surface. (Scale represents 1 mm=1.76 μm.) FIG. 3( b) is atransmission electron microphotograph of blank zein nanoparticles.(Scale represents 1 mm=8.038 nm.) FIG. 3( c) is a scanning electronmicrophotograph of coumarin-loaded zein nanoparticles. (Scale represents1 mm=0.87 μm). FIG. 3( d) is a transmission electron microphotograph of6,7 hydroxy coumarin-loaded zein nanoparticles. (Scale represents 1mm=8.04 nm.)

FIG. 4 depicts atomic force microscopy (AFM) images of blank zeinnanoparticles produced in accordance with the methods of the inventionin the tapping mode in air. Left to right are height, amplitude, andphase images of a representative sample with z-scale of 14.19 nm, 22.2V, and 45°, respectively. The scan size is a 1.14×1.14 μm. The averageparticle size among 50 particles measured in AFM is 185 nm.

FIG. 5 is a graph illustrating the influence of buffer type on theparticle size of coumarin-loaded zein nanoparticles made in accordancewith the methods of the invention before and after lyophilization. Useof citrate buffer in the precipitation method of the present inventionproduces consistently smaller sizes of nanoparticles followinglyophilization as compared with the use of phosphate buffer. (* p<0.05).Each point on the graph represents the mean+SD (n=3). Citrate buffer wascomposed of citric acid (0.0153 g/L) and sodium citrate (2.91 g/L) indeionized water. Phosphate buffer was composed of dibasic sodiumphosphate (1.44 g/L), monobasic potassium phosphate (0.25 g/L) andsodium chloride (10 g/L) in deionized water. Both buffers were used tomaintain the second aqueous phase at pH 7.4 in accordance with theinvention.

FIG. 6 illustrates by means of a flow chart the general steps of themethod of the present invention for preparing doxorubicin loadednanoparticles.

FIG. 7 illustrates an in vitro release profile of 6, 7 hydroxycoumarin-loaded zein nanoparticles in phosphate buffered saline (pH7.4). Coumarin-loaded zein nanoparticles (10 mg/ml) prepared by themethod of the present invention were placed in a dialysis membrane(Spectrapor™, M.wt. 5000 Da) and incubated in phosphate buffered saline(pH 7.4) in the absence (non-enzymatic) or presence (enzymatic) oftrypsin (10 mg/ml). Ethanol (20% v/v) was added to the media to maintainsink conditions, and sodium azide (0.005% w/v) was used as ananti-microbial agent. The solution was maintained at 37° C. in ahorizontal shaker waterbath at 50 rpm. An aliquot (1 ml) of thedialysate was removed at different time points for 7 days and replacedwith fresh media to maintain the sink conditions. Dialysate was analyzedfor coumarin released from the zein nanoparticles usingspectrofluorimetry (λ_(ex)=490 nm; λ_(em)=520 nm). Each data point is amean of three experiments (±SD). Enzymatic release was higher comparedto non-enymatic release at all time points (p<0.05).

FIG. 8 illustrates the in vitro release profile of doxorubicin from zeinnanoparticles in phosphate buffered saline (pH 7.4). Doxorubicin-loadedzein nanoparticles (10 mg/ml) prepared by the nanopreciptation method ofthe present invention were incubated in 1 ml of phosphate bufferedsaline (pH 7.4) in a centrifuge tube and the solution was maintained at37° C. in a horizontal shaker water bath at 50 rpm. The sample wascentrifuged at 10,000 rpm for 10 minutes and the supernatant wasanalyzed for doxorubicin released from the nanoparticles using HPLC. AC-18 column was used and the mobile phase (flow rate 1 ml/min) was 0.1%TFA: Acetonitrile (acetonitrile gradient from 5 to 80% was used). Afluorescence detector (λ_(ex)=505 nm; λ_(em)=550 nm) was used to detectdoxorubicin. The release study was conducted for up to four days. Eachdata point is a mean of three experiments (±SD).

FIG. 9 illustrates by means of a flow chart the general steps of amethod of the present invention for the preparation of dextran-FITC(fluoroisothiocyanate)-loaded nanoparticles. The molecular weight ofdextran is 4000 Da.

FIG. 10 illustrates an in vitro release profile of dextran-FITC fromzein nanoparticles in phosphate buffered saline (pH 7.4).Dextran-FITC-loaded zein nanoparticles (10 mg/ml) were prepared by amethod of the present invention, were incubated in 1 ml of phosphatebuffered saline (pH 7.4) in a centrifuge tube and were maintained at 37°C. in a horizontal shaker water bath at 50 rpm. The sample wascentrifuged at 10,000 rpm for 10 minutes and the supernatant wasanalyzed for dextran-FITC released from the nanoparticles by use ofspectrofluorimetry (λ_(ex)=490 nm; λ_(em)=520 nm). The study wasconducted for eight days. Each point represents the mean±SD (n=3).

FIG. 11 illustrates by means of a flow chart the general steps of themethod of the present invention for the preparation of plasmidDNA-loaded nanoparticles. The plasmid DNA (pDNA) encoding for greenfluorescent protein (GFP) that was used in the study was propagatedusing a DH5α strain of E. coli, which was grown in LB medium. Theplasmid was isolated using Qiagen's ‘EndoFree Plasmid Mega Kit’. Thepurified pDNA was characterized using UV-spectrophotometer, bycalculating the ratio of UV absorbance at 260/280 nm, and alsocharacterized by agarose gel electrophoresis.

FIG. 12 illustrates the influence of particle size on uptake of zeinnanoparticles by porcine polymorpho-nuclear cells. The figure shows thepercent area under the curve for luminal chemiluminescence (over 90minutes) in the presence of zein particles and positive control zymosan.Each experiment is an average of four experiments (±SEM). Uptake issignificantly low in smaller particle sized (p<0.05) compared to othergroups.

FIG. 13 illustrates anti-zein antibodies (optical density) measuredafter the third and fifth weeks of primary and booster subcutaneousinjections of zein particles, respectively. Each value is represented asmean±SEM (n=4). Both the primary and booster titres were statisticallynot significant (p>0.05) compared to the saline group. A coarse zeinsuspension or zein particles in saline (equivalent to 100 μg/50 μl )were injected subcutaneously in female Balb/c mice. Blood was withdrawnfrom the orbital plexus and the anti-zein antibody levels in the dilutedserum (1/16) were measured using a mouse ELISA kit.

FIG. 14 is a graph illustrating the influence of yellow zein (Y) andwhite zein (W) on cell viability of porcine intestinal epithelial cells(IPEC-J2 cells) (at 20,000 cells/well) expressed as the relativeactivities of mitochondrial dehydrogenase after four hours of treatmentusing a dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT)assay. The plate without any treatment was used as a control and wasconsidered to be 100% viable. Zein powder was dissolved in 55% v/vethanol and subsequent dilutions were made from 5 mg/ml stock inserum-free media. At all concentrations, both yellow and white zein donot differ significantly from the control with no treatment (* p<0.05).Each data point is an average of three experiments ±SEM.

FIG. 15 illustrates an in vitro cytotoxicity profile of doxorubicinsolution and doxorubicin-zein nanoparticles prepared in accordance withthe invention in OVACAR-3 cells (human ovarian cancer cells). Cells wereexposed to doxorubicin solution and doxorubicin-loaded nanoparticles ata concentration of 0.0001 to 10 μM for 24 hours. The drug treatment wasremoved after 24 hours and the cells were incubated with blank medium(medium changed every 48 hours) for five days, and the cell viabilitywas measured on the fifth day by MTT assay. Each data point is anaverage of four experiments. The IC₅₀ for doxorubicin solution anddoxorubicin-zein nanoparticles were 3.1 nM and 0.20 nM, respectively.(Dox=doxorubicin solution; Dox-NP=doxorubicin nanoparticles.)

FIG. 16 illustrates an in vitro cytotoxicity profile of doxorubicinsolution and doxorubicin-zein nanoparticles prepared in accordance withthe invention in doxorubicin-resistant human breast cancer cells(NCI/ADR-RES cells). Cells were exposed to doxorubicin solution anddoxorubicin-loaded nanoparticles at a concentration of 0.0001 to 10 μMfor 24 hours. The drug treatment was removed after 24 hours and thecells were incubated with blank medium (medium changed every 48 hours)for five days, and the cell viability was measured on the fifth day byan MTT assay. Each data point is an average of four experiments. TheIC₅₀ for doxorubicin solution and doxorubicin-loaded nanoparticles were81.73 nM and 6.41 nM, respectively. (Dox=doxorubicin solution;Dox-NP=doxorubicin-loaded nanoparticles.)

FIG. 17 illustrates by means of a flow chart a method of the presentinvention for preparing cross-linked blank zein nanoparticles.

FIG. 18 is a graph demonstrating the extent of cross-linking of zeinnanoparticles as a function of cross-linking agent for 24 hrs. Theextent of cross-linking was determined using a TNBS assay. Thecross-linking agents used were GTA—Glutaraldehyde (500 μl of a stocksolution of 25% w/v), EDC: 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide(0.6% w/v), and NHS: N-hydroxyl succinimide (0.6% w/v). Theconcentration of genipin used was 0.05% w/v. “Blank” represents zeinnanoparticles without any cross-linking agent. Data is a mean of twoexperiments.

FIG. 19 illustrates by means of a flow chart a method of the presentinvention for preparing rhodamine-123-loaded cross-linked zeinnanoparticles.

FIG. 20 illustrates the in vitro release profile of rhodamine-123 fromzein nanoparticles in phosphate buffered saline (0.1 M) pH 7.4. Resultsrepresent mean±SEM (n=4). NCS=non-cross linked particles; CS=crosslinked particles. The drug release from cross-linked nanoparticles wassignificantly (p>0.05) lower than the non cross-linked nanoparticles.Rhodamine-loaded zein nanoparticles (20 mg) prepared by the method ofthe present invention were placed in a dialysis membrane (Spectrapor™,M.wt. 10,000 Da) and incubated in 5 ml of phosphate buffered saline (pH7.4). The solution was maintained at 37° C. in a horizontal shaker waterbath at 100 rpm. An aliquot (1 ml) of the dialysate was removed atdifferent time points over 48 hours and replaced with fresh media tomaintain the sink conditions. Dialysate was analyzed for rhodaminerelease from the zein nanoparticles using spectrofluorimetry (λ_(ex)=485nm; λ_(em)=530 nm).

FIG. 21 illustrates the in vitro release profile of rhodamine-123 fromzein nanoparticles in the presence of trypsin at pH 7.4. Resultsrepresent mean±SEM (n=4). ENCS=non-cross linked particles; ECS=crosslinked particles. The drug release from cross-linked nanoparticles wassignificantly (p>0.05) lower than the non cross-linked nanoparticles.Rhodamine-123-loaded zein nanoparticles (20 mg) prepared by the methodof the present invention were placed in a dialysis membrane(Spectrapor™, M.wt. 10,000 Da) and incubated in 5 ml of phosphatebuffered saline (0.1M, pH 7.4) containing 205 μg/ml of trypsin. Thesolution was maintained at 37° C. in a horizontal shaker water bath at100 rpm. An aliquot (1 ml) of the dialysate was removed at differenttime points over 48 hours and replaced with fresh media to maintain thesink conditions. Dialysate was analyzed for rhodamin-123 released fromthe zein nanoparticles using spectrofluorimetry (λ_(ex)=485 nm;λ_(em)=530 nm).

FIG. 22 illustrates in a flow chart the general method for preparationof blank PEGylated zein nanoparticles.

FIG. 23 is a graph illustrating an intensity of weighted sizedistribution of PEGylated nanoparticles. The particle size of PEGylatedzein nanoparticles was 131±1 nm, with a Polydispersity Index (PDI) of0.282±0.01. The data also shows that the surface modification of zeinnanoparticles with PEG does not increase the particle size and that thenanoparticles are in the desired size range for drug deliveryapplications.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the term “nanoparticle” is generally known to refer to aparticle that is not more than 1000 nm in at least one dimension.However, the nanoparticles formed by the methods of the presentinvention will have a diameter of a specified value as defined herein.Further, the use of the term “nanoparticle” is also meant to refergenerically to blank nanoparticles and nanoparticles loaded with amolecule and formed by methods of the present invention.

As used herein, unless defined otherwise (i.e., FIG. 18), “blanknanoparticle” refers to and means nanoparticles formed in accordancewith the methods of the invention that do not have a selected particle,molecule or material formed with or in conjugation with thenanoparticle.

As used herein, the term “diameter,” when used in the context ofnanoparticle dimensions, refers to the mean linear dimension of theparticle for lines passing through the center of mass of the particle.Acceptable approximation of the diameter of non-spherical particles maybe provided, for example, by taking the mean of the thickness of theparticle along three orthogonal axes of a coordinate system, with one ofthe axes aligned with the longest dimension of the particle.

As used herein, the term “administered” or “administration,” when usedin the context of therapeutic and diagnostic uses for nanoparticles,refers to and includes the introduction of a selected amount ofnanoparticles into an in vivo or in vitro environment for the purposeof, for example, delivering a therapeutic agent to a targeted site.

As used herein, “in vivo” means of or within the body of a subject, suchas that of a patient, and includes administration of nanoparticles by avariety of means including, but not limited to, oral, intravenous,intraperitoneal, parenteral, subcutaneous, topical, opthomogical andnasal routes of administration.

As used herein, “in vitro” means or refers to environments outside ofthe body of a subject or patient.

As used herein, the terms “subject” or “patient” both refer to or meanan individual complex organism, e.g., a human or non-human animal.

As used herein, “grades of zein” refers to a variety of types or formsof zein, including white zein and yellow zein, derived by various means,such as is disclosed in U.S. Pat. No. 5,254,673, the contents of whichare incorporated herein [9].

As used herein, the term “therapeutic agent,” and similar termsreferring to a therapeutic or medicinal function mean that thereferenced molecule, macromolecule, drug or other substance canbeneficially affect the initiation, course, and/or one or more symptomsof a disease or condition in a subject, and may be used in conjunctionwith nanoparticles in the manufacture of medicaments for treating adisease or other condition.

As used herein, the term “biocompatible” means that the nanoparticleproduced by the disclosed method of the invention does not cause orelicit significant adverse effects when administered in vivo to asubject. Examples of possible adverse effects include, but are notlimited to, excessive inflammation and/or an excessive or adverse immuneresponse, as well as toxicity.

As used herein and in the appended claims, the singular forms, forexample, “a”, “an”, and “the,” include the plural, unless the contextclearly dictates otherwise. For example, reference to “a nanoparticle”includes a plurality of such nanoparticles, and reference to a“molecule” is a reference to a plurality of molecules, and equivalentsthereof.

As used herein, “about” or “approximately” means reasonably close, to ora little more or less than, the stated number or amount.

As used herein, “comprising,” “including,” “having,” “containing,”“characterized by,” and grammatical equivalents thereof, are inclusiveor open-ended terms that do not exclude additional, unrecited elementsor method steps, but also include the more restrictive terms “consistingof” and “consisting essentially of.”

The present invention relates to methods of producing non-immunogenicnanoparticles from hydrophobic water-insoluble proteins by controllingthe particle size of the nanoparticles within a size range ofapproximately 100 nm to 400 nm, and most suitably within a size range ofbetween approximately 100 nm and 300 nm. FIG. 1 illustrates by means ofa flow chart the general steps of preparing non-immunogenicnanoparticles by the method of the present invention.

In an initial step or phase of the method, a water-insoluble protein(0.4 to 1.25% w/v) is dissolved in a hydroalcoholic solvent that maycontain ethanol and deionized water. The composition of the solvent maybe 90:10% v/v or 92:8% v/v, for example. For methods where a selectedmolecule is to be encapsulated in the nanoparticle, the molecule (0.03to 0.3% w/v) to be encapsulated is added to the solution of this firstaqueous phase. The molecule to be encapsulated is approximately 5 to 50%w/w of the protein polymer.

The pH of the solution may be altered to bring the pH of the solution tobetween about pH 6 and about pH 7 by the addition of 0.01 NaOH or 0.01NHC. If the water pH changes after addition of an acidic molecule, suchas coumarin, or by a basic molecule, the pH is to be adjusted to pH 6 to7. The solution of the first phase may be processed by probe sonicationto aid is the dissolution of the protein.

In a subsequent step of the method, the aqueous solution of the initialstep or phase is added to a buffering agent under ultrasonic shear.Citrate buffer is particularly preferred. The choice of the bufferingagent utilized for the second aqueous phase is considered to besignificant for maintaining the pH during nanoparticle formation, and isalso significant for subsequent lyophilization of the formednanoparticles as described later in this disclosure. If no buffer isused, or if, for example, 0.1N HCl is used to adjust the pH of thesecond aqueous phase solution, the particles produced tend to be largerthan those produced with the citrate buffer, and the particles tend todemonstrate a wider size range. Use of a citrate buffer produces some ofthe smallest particle diameter sizes, such as approximately 100 nm. Useof other buffers may produce particles in the same or similar diametersize range of approximately 100 nm to approximately 300 nm, but afterthe lyophilization step, the size of the nanoparticles formed usingother buffering agents tends to increase by two to three times.

Significantly, the pH of the second aqueous phase solution is preferablybetween approximately pH 6.8 and approximately pH 7.4 to obtain thedesired size of nanoparticles. If the pH is outside of this range, theparticle size tends to become larger, and the polydispersity index (PDI)of the particles produced is higher. The PDI is a measure of thedistribution of the particles in different size ranges. The method thusmay utilize the solubility difference of a protein, such as zein, in thehydroalcoholic solution and an aqueous solution with a selected pH ofapproximately 6.8 to approximately 7.4 close to the isoelectric point ofzein.

Further, the addition of a buffering agent to the second aqueous phasesolution may be performed under high ultrasonic shear or under highpressure homogenization, or a combination of both ultrasonic shear andhigh pressure homogenization. The ultrasonic energy and duration ofultrasonic shear may be particularly significant to the formation ofparticles in the desired diameter size ranges. The ultrasonic shearenergy may be carried out from 0.6 kW/h to 1.39 kW/h, for a duration ofapproximately 2 to 10 minutes with a pulse on-time of from 5 to 10seconds and an off-time of from 1 to 5 seconds. The ultrasonicprocessing may be significant to the production of particles in thedesired size range. When employing high pressure homozenization, theprocess may be carried out using an orifice size of between 0.1 mm and0.25 mm, and for a time period of between five to ten minutes at apressure of from 5000 to 40,000 psi.

The buffering agent of the second phase may also preferably contain asurfactant and a phospholipid in a selected ratio. The ratio ofsurfactant to phospholipid may be approximately 2:1 w/w, which isbelieved to produce the most desirable results. The ratio may also be1:0.5% w/w or 1:1% w/w or 1:2% w/w. Significantly, the utilization ofthe combination of a surfactant and a phospholipid is highly desirableto stabilize the particles produced and to help prevent aggregations ofthe particles. By way of example only, the surfactant may be apoloxamer, such as Pluronic® F68, and the phospholipid may be lecithin.Other surfactants that may be used in the method include other nonionicsurfactants such as poloxamers (Pluronic®), polyoxyethylene alkyl ethers(Brij), sorbitan esters (Span), polyoxyethylene sorbitan fatty acidesters (Tween), and ionic surfactants such as sodium dioctylsulfosuccinate, sodium lauryl sulfate, benzalkonium chloride, cetyltrimethyl ammonium bromide, n-dodecyl trimethyl ammonium bromide, andpolymer such as polyvinyl alcohol, polyvinyl pyrrolidone. Otherphospholipids that may be used in the method include nonionic andcharged lipids or phospholipids such as egg lecithin, soy lecithin,phosphatidyl choline, phosphatidyl ethanolamine,1,2-dioleoyl-3-trimethyl ammonium propane.

A combination of poloxamer and lecithin (e.g., 0.9% w/w:0.45% w/w) inthe selected ratio has been found to produce nanoparticles in thedesired diameter size range of approximately 100 nm to approximately 300nm. Use of either of the surfactant or phospholipid alone has generallybeen found to result in larger particle sizes outside of the desireddiameter size range. However, the use of either a surfactant or aphospholipid in accordance with the methods disclosed herein will resultin nanoparticles of a desired size for non-immunogenicity.

After the application of ultrasonic shear or/or high pressurehomozenization to the solution of the second phase, the mixture may bestirred to evaporate the ethanol or other solvent to form thenanoparticles. The stirring may be performed by a mechanical stirrer,and may be performed at a rate of from approximately 300 rpm toapproximately 500 rpm at room temperature for approximately three hours.

The nanoparticles may preferably then be subjected to ultracentrifugalfiltration for the purpose of separating the nanoparticles from theresidual material. Ultracentifugation may be carried out usingcentrifugal filters of molecular weight cut-off of about 5000 Da (orother appropriate filters with a higher or lower Mwt cut-off than 5000Da), and at between 2000 g and 40,000 g, depending on the encapsulatedmolecule or drug, or on the particular treatment of the nanoparticles,such as PEGylation. The time of the ultracentrifugation can vary frombetween 20 and 50 minutes.

A cryoprotectant may then be added to the nanoparticles. For example, 2%w/v trehalose may be added as a cryoprotectant. Other cryo- orlyoprotectants can also be used, such as sugars, including glucose,sucrose, lactose, ficoll, betaine or mannitol or polyols such asmannitol, sorbitol, which can be used as lyoprotectants. Thenanoparticles may be kept at −80° C. to form a solid cake, which is thenlyophilized, such as by drying the nanoparticles in a frozen state underhigh vacuum. The duration of ultrasonic energy, type of surfactant,concentration of surfactants, and buffer may be varied.

By way of example, nanoparticles having a size range distribution ofbetween approximately 100 nm and approximately 400 nm were prepared asfollows:

Example I

In a first aqueous phase, 0.0135 g of white zein was dissolved in amixture of 3 ml of ethanol and 0.25 ml of water. The concentration ofzein or solvent combination used was optimal; however, nanoparticles inthe desired different size range can be produced by modifying the zeinconcentration or solvent composition. Dissolution of the zein was aidedby the application of probe sonication for about 20 seconds. Theresulting solution of the first aqueous phase was then added drop-wiseinto a 15 ml solution of citrate buffer, with a pH 7.4, and acombination of lecithin (0.45% w/v) and Pluronic® F68 (0.9% w/v) underconstant application of ultrasonic energy (1.39 kW/h, 37% amplitude) for10 minutes with a pulse on time of 10 seconds and off time of 1 second.During the ultrasonic shearing process, the dispersion was kept in anice bath to maintain the temperature at about 10° C. The dispersion wasthen placed on a magnetic stirrer at between 300 to 500 rpm, at roomtemperature, until the ethanol was completely evaporated. After completeevaporation of the ethanol, the nanoparticles were purified to removeany residual materials and/or surface active agents. Purification wasaccomplished by repeated washing with deionized pH 7.4 citrate bufferand ultracentrifugation using centrifugal filters of MWt cut off of 5000Da, at 3950 g for 50 minutes. To 4 ml of the resulting aqueoussuspension (pH 7.4 citrate buffer) of zein nanoparticles was added 2%w/v trehalose as a cryoprotectant, and the nanoparticles were then keptat −80° C. to form to a solid cake. The material was then lyophilized at−47° C. and at 60 mTorr vacuum for 12 to 14 hrs. The nanoparticles werethen stored in a refrigerator at 10° C. in a dessicator.

In an alternative method of the invention, the ultrasonic shear of thesecond phase solution can be supplemented or replaced by high pressurehomogenizer by passing the dispersion under high pressure through anarrow orifice for reducing the particle size. This is especially usefulto produce nanoparticles in the smaller size range when a highconcentration of zein is used. Also high pressure homogenization can beused as a scale-up method for preparing zein nanoparticles. An exampleof the method is described below.

Example II

An amount of 0.65% w/v white zein was dissolved in a mixture of 6 ml ofethanol and 0.50 ml of water. The composition of the resulting solutionof the first aqueous phase was altered to obtain a desired pH of aboutpH 6 to about pH 7. Dissolution of the zein was aided by the applicationof probe sonication for about 20 seconds. The resulting solution of thefirst aqueous phase was then added drop-wise into a 30 ml solution ofcitrate buffer, having a pH 7.4, and a combination of lecithin (0.45%w/v) and Pluronic® F68 (0.9% w/v) under constant application ofultrasonic energy (1.39 kW/h, 37% amplitude) for 2 minutes with a pulseon time of 10 seconds and off time of 1 second. During the ultrasonicshearing process, the dispersion was kept in an ice bath to maintain thetemperature at about 10° C. The resulting coarse suspension was thenpassed through a high pressure homogenizer (Nano Debee®, USA) having anorifice size of between 0.1 and 0.25 mm for five minutes at 20,000 psi.During the high pressure homogenization process the temperature of ismaintained at approximately 10° C. by circulating water in the highpressure homogenizer using a chiller. Subsequently, the dispersion waskept on a magnetic stirrer at 300 to 500 r.p.m and at room temperatureuntil the ethanol was completely evaporated. After complete evaporation,the nanoparticles were purified to remove any residual materials orsurface active agents. Purification was accomplished by repeated washingwith pH 7.4 citrate buffer and ultracentrifugation using centrifugalfilters of MWt cut off of 5000 Da, at 3950 g for 50 minutes. Fourmilliliters of aqueous suspension (pH 7.4 citrate buffer) ofnanoparticles was mixed with 35 mg of 2% w/v trehalose, and was kept at−80° C. to form a solid cake. The cake was then lyophilized at −47° C.and 60 mTorr vacuum for 12 to 14 hrs.

The methods of the invention described in Examples I and II can beadapted for the formation of nanoparticles where a selected molecule,such as a therapeutic drug, is encapsulated within a nanoparticle (FIG.2). An example of a method of the invention for forming amolecule-encapsulated nanoparticle is as follows:

Example III

White zein in the amount of 0.0135 g was dissolved in a mixture of 3 mlethanol and 0.25 ml of 0.01 N NaOH to adjust the pH between 6 and 7. Tothe solution was added 0.0066 g of 6,7-hydroxy coumarin and the mixturewas subjected to probe sonication for 20 seconds to assure dissolution.The resulting solution was added drop-wise into 15 ml of citrate buffer(pH 7.4) containing 0.0675 g of lecithin and 0.135 g of Pluronic® F68under constant ultrasonic energy at 1.39 kW/h and 37% amplitude for 10minutes, with a pulse on-time of 10 seconds and an off-time of 1 second.During the sonication process, the solution was kept in an ice bath tomaintain the temperature around 10° C. Subsequently, the dispersion wasplaced on a magnetic stirrer at 300 to 500 r.p.m and at room temperatureuntil the ethanol was completely evaporated. Following completeevaporation of the alcohol, the nanoparticles were purified to removeany excess drug and/or surface active agents. Purification wasaccomplished by repeated washing with pH 7.4 citrate buffer andultracentrifugation using a centrifugal filter of MWt cut off of 5000Da, at 3950 g for 50 minutes. Four milliliters of the aqueous suspension(pH 7.4 citrate buffer) of coumarin-loaded nanoparticles were added with35 mg of trehalose and was kept at −80° C. to form a solid cake. Thesolid cake was then lyophilized at −47° C. and 60 mTorr vacuum for 12 to14 hrs.

It has been shown that white zein may be suitably used in the methods ofthe present invention as the base protein. White zein gives reproduciblenanoparticles in a desired narrow size range of approximately 100 nm toapproximately 400 nm, while yellow zein gives larger particles withwider particle size distribution. This difference is illustrated inTable 1 and Table 2, below. Table 1 provides data of nanoparticles madefrom yellow zein by the method of Example I and Example III, above. Bothblank and coumarin-loaded nanoparticles are shown. It can be seen thatthe particle size of each is approximately 460 nm and 610 nm,respectively. By comparison, as shown in Table 2, below, blank andcoumarin-loaded nanoparticles made from white zein by the method ofExample I and Example III are smaller. FIGS. 3 and 4 show electronmicroscopic and atomic force image of the blank and coumarin-loaded zeinnanoparticles.

TABLE 1 Particle Polydispersity Zeta Model Size index PotentialEncapsulation compound (nm) (PDI) (mV) Efficiency (%) Blank zein 460 ±63  0.46 ± 0.06 −10.28 ± 2 Not applicable nanoparticles 6,7 Hydroxy 610± 123 0.62 ± 0.08 −16.28 ± 3 98 ± 1.5 coumarin Each value is an averageof three experiments with ±SD.

TABLE 2 Particle Polydispersity Zeta Model Size index PotentialEncapsulation compound (nm) (PI) (mV) Efficiency (%) Blank zein 224 ± 200.31 ± 0.06  −16 ± 3 Not applicable nanoparticles 6,7 Hydroxy 266 ± 300.44 ± 0.08 −11.34 ± 1.8 62 ± 17 coumarin Each value is an average ofthree experiments with ±SD.

The pigments in yellow zein appear to affect the solubility of zein andthe formation of nanoparticles of the desired size distribution. It hasbeen found in the prior art to be particularly challenging to prepareparticles using natural polymers, such as proteins, that areconsistently within the desired size range. However, the presentinvention can produce nanoparticles consistently in the desired sizerange using a suitable grade of protein, such as white zein.

Significantly, the methods of the invention may produce, and haveproduced, nanoparticles with a diameter size as low as 80 nm to 100 nm.If part of the ultrasonic shear is replaced by high pressurehomogenization, as described in Example II, above, the resultingparticle size of blank nanoparticles is also similar to the particlesizes shown in Table 2, above, namely having a particle size ofapproximately 220±15 nm and a PDI of 0.4±0.07.

The yield of nanoparticles produced by the nanoprecipitation methods ofthe present invention that are in the desired size range has been foundto be greater than approximately 60%. The methods are significant inthat the particles produced have diameters that primarily measure in arange of less than approximately 400 nm, and preferably with arelatively narrow diameter size distribution of approximately 100 nm toapproximately 300 nm to avoid an immunogenic reaction when administeredinto the body. Advantageously, zein nanoparticles in the diameter sizerange of approximately 100 to approximately 400 nm, such as are producedby the methods of the invention, are not taken up by phagocytic cells,while larger particles of a diameter size greater than approximately 400nm are rapidly taken up by phagocytic cells when tested in vitro usingporcine blood. This suggests that nanoparticle phagocytosis is avoidedby controlling the particle diameter size of zein nanoparticles in thesmaller size range.

Immunogenicity studies in mice showed that zein nanoparticles in thediameter size range of approximately 100 to approximately 400 nm arenon-immunogenic, while zein nanoparticles having a diameter size greaterthan approximately 400 nm produced a significant immune response(anti-zein antibodies were two- to four-fold higher compared to salinecontrol). These results show that preparing and using nanoparticleshaving diameter sizes less than approximately 400 nm helps avoid anysignificant immunogenicity caused by the hydrophobic proteins of theparticles.

The ability to control size of the nanoparticles is achieved in part bycontrolling the pH of the solution in the second aqueous phase of themethod. The data in Table 3, below, illustrates that smaller sizes ofnanoparticles, with a lower PDI, are achieved at a pH of between 6.8 and7.4.

TABLE 3 pH of the aqueous phase Particle Size (nm) Polydispersity index1.5 362 ± 24 0.392 3 291 ± 15 0.45 6.8 208 ± 10 0.289 7.4 232 ± 7  0.26010 256 ± 20 0.317 12 368 ± 10 0.438 Each value is an average of threeexperiments with ±SD

A further critical factor in controlling the size of nanoparticleformation is the combination of surfactant and phospholipids which isrequired to stabilize the nanoparticles and prevent particleaggregation. A combination of a poloxamer and lecithin, such as in a 2:1ratio (e.g., 0.9:0.45% w/w), produces nanoparticles in the desired sizerange. If either the surfactant or the phospholipid is used alone,larger particles are obtained, as suggested by the data of Table 4,below.

TABLE 4 Surfactant (% w/v) Particle size (nm) PDI Pluronic ® (0.9) 516 ±75 0.57 ± 0.07 Lecithin (0.9)* 335 ± 45 0.52 ± 0.05 Pluronic ® (0.9) andLecithin (0.4) 274 ± 36 0.46 ± 0.02 Each value is an average of threeexperiments with ±SD. *Lyophilization resulted in a sticky powder.

The choice of buffering agent for the second aqueous phase is not onlycritical to maintaining the optimum pH during nanoparticle formation,but is also critical for subsequent lyophilization. For example, if nobuffering agent is used in the second aqueous phase solution, or if 0.1NHCl is used to adjust the pH, the resulting nanoparticles are larger insize, with a wider size range or PDI. As shown in FIG. 5, the use ofcitrate buffer gave the smallest particle size (109±12 nm). The use ofother buffering agents, particularly phosphate, results in the particlesize of zein nanoparticles being increased by two to three times afterlyophilization.

The graph of FIG. 5 illustrates that zein nanoparticles prepared by themethod using phosphate as the buffering agent in the solution from thesecond aqueous phase and obtained after lyophilization produced muchlarger particles as compared to nanoparticles prepared using citratebuffer as the buffering agent in the second aqueous phase. The particlesize increase in phosphate buffer is probably due to the crystallizationand precipitation of buffer at the freeze-drying temperatures caused bythe pH drop [10]. This problem is solved using citrate buffer, whicheffectively resists the changes in pH during freeze-drying temperatures.The amino groups in zein can be cross-linked by citric acid and thisalso stabilizes the zein nanoparticles [11].

It is notable that zein is a biodegradable protein and is also morebiocompatible than synthetic polymers. Zein is a polymer that is listedas a GRAS (Generally Regarded As Safe) polymer by FDA standards [12].The method of the invention is, therefore, suitable for preparing zeinnanoparticles with encapsulated molecules or drugs of differentphysiochemical properties. Table 5, below, illustrates by way of examplea sampling of some molecules that may be encapsulated by nanoparticlesusing the methods in accordance with the present invention. The numberor type of molecules that may be used in the nanoparticle encapsulationare not limited to those noted herein.

TABLE 5 Particle Size Zeta Encapsulation Model compound (nm) potentialefficiency (%) 6,7-hydroxy coumarin 173 ± 20 −16 ± 3 68 ± 6 Doxorubicin171 ± 45 −21 ± 2  61 ± 16 Dextran FITC (4000 Da)  89 ± 12 −15 ± 2 79 ± 8pDNA (GFP) 185 ± 12  −17 ± 0.4 86.2 ± 3  Each value is a mean of threeexperiments with ±SD.

An example of a nanoparticle formed with 6, 7 hydroxy coumarin isdescribed in Example III above and is shown in FIG. 2. Another exampleof a nanoparticle containing a therapeutic agent is doxorubicin-loadedzein nanoparticles, the general steps of which are illustrated in FIG.6. A specific method for preparing doxorubicin-loaded zein nanoparticlesis as follows:

Example IV

White zein in the amount of 0.0135 g was dissolved in a mixture of 3 mlof ethanol and 0.25 ml of water. To this solution of the first aqueousphase was added 0.001 g of doxorubicin hydrochloride and the mixture wasprobe sonicated for 20 seconds to dissolve the doxorubicinhydrochloride. The resulting solution was added drop-wise into 15 ml ofcitrate buffer (pH 7.4) containing 0.0675 g of lecithin and 0.135 g ofPluronic® F68 under constant ultrasonic energy at 1.39 kW/h and 37%amplitude for 10 minutes with a pulse on-time of 10 seconds and off-timeof 1 second. During the sonication process, the solution was kept in anice bath to maintain the temperature at about 10° C. Subsequently, thedispersion was placed on a magnetic stirrer at 300 to 500 r.p.m at roomtemperature until the ethanol was completely evaporated. After completeevaporation of the alcohol, the nanoparticles were purified to removeresidual material. Purification was accomplished by repeated washingwith pH 7.4 citrate buffer and subjected to ultracentrifugation, usingcentrifugal filters of MWt cut off of 5000 Da, at 3950 g for 50 minutes.To the aqueous suspension (pH 7.4 citrate buffer) of doxorubicinnanoparticles was added 35 mg of trehalose and the mixture was kept at−80° C. to form a solid cake. The material was then lyophilized at −47°C. and 60 mTorr vacuum for 12 to 14 hrs.

In preparation of the doxorubicin-loaded zein nanoparticles according tothe method (FIG. 6), particles were formed having a mean diameter ofapproximately 171±45 nm and a PDI of approximately 0.3. Theencapsulation efficiency of doxorubicin by the zein nanoparticles wasapproximately 61±16%.

Zein nanoparticles made in accordance with the present invention providea beneficial and/or advantageous sustained release of the encapsulatedmolecule or drug due in part to the water insolubility of zeinnanoparticles that enable the particles to sustain the drug release overa period of time. For example, FIG. 7 depicts the in vitro releaseprofiles for coumarin-loaded nanoparticles made in accordance with themethod described in Example II, above. The data indicates that in vitro,there is a sustained release of the drug over a period of up to sevendays, with a higher release rate being observed in the presence ofenzymes. The data shows that the zein nanoparticle release is mediatedby slow diffusion of drug out of the nanoparticle and slow enzymaticbreakdown of zein nanoparticles. FIG. 8 depicts the in vitro releaseprofile of doxorubicin from the doxorubicin-loaded zein nanoparticlesmade according to Example IV, showing a mixed order with an initialburst followed by a sustained release after approximately 24 hours.

A further example of a therapeutic or diagnostic agent that may beformed as a nanoparticle in accordance with the invention isDextran-FITC (FIG. 9). An example of preparing a Dextran-FITC-loadedzein nanoparticles is as follows:

Example V

An amount of 0.0135 g of white zein was dissolved in a mixture of 3 mlof ethanol and 0.25 ml water. To the zein solution was added 0.003 g ofdextran (Mwt 4000 Da) labeled with FITC and the dextran-FITC wasdissolved in the above solution. The resulting solution was addeddrop-wise into 15 ml of citrate buffer (pH 7.4) containing 0.0675 g oflecithin and 0.135 g of Pluronic® F68 under constant ultrasonic energyat 1.39 kW/h and 37% amplitude for 10 minutes with a pulse on-time of 10seconds and off-time of 1 second. During the sonication process, thesolution was kept in an ice bath to maintain the temperature at about10° C. Subsequently, the dispersion was placed on a magnetic stirrer at300 to 500 r.p.m at room temperature until the ethanol was completelyevaporated. After complete evaporation of the alcohol solvent, thenanoparticles were purified to remove the residual materials.Purification was accomplished by repeated washing with pH 7.4 citratebuffer and ultracentrifugation, using centrifugal filter of MWt cut offof 5000 Da, at 3950 g for 50 minutes. To the aqueous suspension (pH 7.4citrate buffer) of dextran-FITC-loaded nanoparticles was added 35 mg oftrehalose and the mixture was kept at −80° C. to form a solid cake. Thematerial was then lyophilized at −47° C. and 60 mTorr vacuum for 12 to14 hrs.

Dextran-FITC nanoparticles prepared in accordance with the invention(FIG. 9) shows a sustained in vitro release profile, as shown in FIG.10.

Further in accordance with the present invention, molecules that aresuitable for gene therapies can also be encapsulate in nanoparticles fortherapeutic and diagnostic use, such as, for example, plasmids, DNA,oligonucleotides and siRNA. FIG. 11 illustrates the general method forpreparing a nanoparticle containing a gene-based molecule. A specificexample for making a nanoparticle comprising pDNA (plasmid DNA)encapsulated in a nanoparticle is as follows:

Example VII

An amount of 0.0135 g of white zein was dissolved in a mixture of 3 mlof ethanol and 0.25 ml water. To the zein solution was added 0.187 μg ofpDNA GFP (green fluorescent protein) which was dissolved in the abovezein solution. The resulting solution was added drop-wise into 15 ml ofcitrate buffer (pH 7.4) containing 0.0675 g of lecithin, 0.135 g ofPluronic® F68 and 7.5 mM of CaCl₂ under constant ultrasonic energy at1.39 kW/h and 37% amplitude for 10 minutes with a pulse on-time of 10seconds and off-time of 1 second. During the sonication process, thesolution was kept in an ice bath to maintain the temperature at about10° C. Subsequently, the dispersion was placed on a magnetic stirrer at300 to 500 r.p.m at room temperature until the ethanol was completelyevaporated. After complete evaporation of the alcohol solvent, thenanoparticles were purified by ultracentrifugation using a centrifugalfilter with a Mwt cut-off of 5000 Da and processing at 3950 g for 50minutes to remove excess drug, and surface active agents. Two cycles ofultracentrifugation were conducted and the nanoparticles are washed withwater. To the aqueous suspension of pDNA-loaded nanoparticles was added35 mg of trehalose and the mixture was kept at −80° C. to form a solidcake. The material was then lyophilized at −47° C. and 60 mTorr vacuumfor 12 to 14 hrs.

The drug release profiles for the various encapsulated molecules, asshown in FIGS. 6, 8 and 10 for example, indicate that zein nanoparticlescan be used as a versatile and safe drug delivery vehicle by parenteraland non-parenteral routes of administration including oral, buccal,transdermal, nasal, pulmonary and ocular routes of delivery. Many othermolecules, particles and drugs may be encapsulated as well, includingbut not limited to, vaccines and cosmetic substances (e.g., Minoxidil,Vitamin C, etc.) for therapeutic, diagnostic and aesthetic applicationsor therapies.

Further, due to the relatively smaller size of the nanoparticles formedby the methods of the present invention, molecule-loaded (e.g.,drug-loaded) zein nanoparticles can circulate in the body for prolongedperiods without being recognized and eliminated by phagocytic cells. Thedata of FIG. 12 illustrate that zein nanoparticles in the size range of100-400 nm are not taken up by the blood phagocytic cells, while largerparticles in the size of >400 nm are rapidly taken up by phagocyticcells when tested in vitro using porcine blood. Thus, it can be shownthat phagocytic uptake is avoided by controlling the particle size ofzein nanoparticles in the smaller size range. Immunogenicity studies inmice showed that zein nanoparticles in the size range of 100 nm to 400nm are non-immunogenic. On the other hand, zein nanoparticles having asize >400 nm produced a significant immune response (two- to four-fold)compared to the control, as shown in FIG. 13.

The cytotoxic effects of the zein used for making the nanoparticles wereinvestigated in cell proliferation studies using porcine intestinalepithelial cells (IPEC-J2). The results of an exemplary cytotoxicitystudies is shown in FIG. 14. No significant degree of cytotoxicity wasobserved between white zein and yellow zein, as compared to controltreatment with buffer at any concentration.

The therapeutic activity of zein nanoparticles made in accordance withthe disclosed methods was tested in vitro using doxorubicin-loaded zeinnanoparticles with human ovarian cancer cells (OVCAR-3) (FIG. 15) anddoxorubicin-resistant human breast cancer cells (NCI/ADR-RES) (FIG. 16).The cells were plated at a seeding density of 2000 cells/well/0.1 ml.Following overnight attachment, the cells were treated with 0.07 to 70nM (OVCAR-3) and 0.1 to 10000 nM (NCI/ADR-RES) concentrations of eitherdoxorubicin solution or doxorubicin-nanoparticles for 24 hours. After 24hours, the respective drug treatments were removed. The cells werewashed twice with ice cold phosphate buffer and replaced with freshmedia. Media was replaced every 48 hrs. An MTT assay was used to assesscytotoxicity on the fifth day following treatment (NCI and OVCAR-3). Theresults show that the doxorubicin-loaded in zein nanoparticles had asignificantly higher potency than the free doxorubicin solution in humancancer cells. Doxorubicin-loaded nanoparticles were approximately 12 to16 times more potent than the free doxorubicin. The difference inpotency is believed to be due to the difference in the cell uptakemechanism of free drug and drug encapsulated in nanoparticles. Freedoxorubicin is taken up by passive diffusion dictated by theconcentration gradient, while the doxorubicin-loaded nanoparticles aretaken up in an active endocytosis process. Further it is believed thatthe endocytosis of doxorubicin-loaded nanoparticles can avoid the drugefflux pumps in resistant cancer cells, thus resulting in betterefficacy.

In a further aspect of the present invention, the enzymatic stability ofthe nanoparticles produced by the disclosed methods of the invention canbe further enhanced by cross-linking FIG. 17 illustrates the generalmethod for preparation of cross-linked blank zein nanoparticles usingglutaraldehyde as the cross-linking agent. A specific example of suchpreparation is as follows:

Example VIII

Blank zein nanoparticles were prepared using the disclosednanoprecipitation method. A cross linking agent was added followingprobe sonication of the second aqueous phase. Nanoparticles were furtherincubated for 24 hours. At the end of incubation time, the nanoparticleswere purified using centrifugal filtration and were then lyophilized.

White zein in the amount of 0.0135 g was dissolved in a mixture of 3 mlof ethanol and 0.25 ml of water. The first phase solution was then addeddrop-wise into 15 ml of citrate buffer having a pH 7.4 and containing acombination of 0.45% w/v lecithin and Pluronic® F68 (0.9% w/v) underconstant application of ultrasonic energy at 1.39 kW/h and 37% amplitudefor 10 minutes with a pulse on-time of 10 seconds and off-time of 1second. During the sonication process, the solution was kept in an icebath to maintain the temperature at about 10° C. To the solution wasadded 0.5 ml of glutaraldehyde of 25% w/v and the solution was incubatedfor 3 to 24 hrs at 37° C. while stirring at 300 to 500 rpm. The residualglutaraldehyde was neutralized with 10% w/v metabisulfite. Subsequently,the dispersion was placed on a magnetic stirrer at 300 to 500 rpm and atroom temperature until the ethanol was completely evaporated. Aftercomplete evaporation of the alcohol, the nanoparticles were purified toremove the residual material. Purification was accomplished by repeatedwashing with pH 7.4 citrate buffer and ultracentrifugation, usingcentrifugal filter of MWt cut off of 5000 Da, at 3950 g for 50 minutes.To the aqueous suspension of nanoparticles was added 35 mg of trehaloseand the solution was kept at −80° C. to form a solid cake. The materialwas then lyophilized at −47° C. and 60 mTorr vacuum for 12 to 14 hrs.

Notably, for other cross-linking agents such as EDC/NHS and genipin,when used in the method of FIG. 13, the reaction time can vary from 24to 72 hours. The surface amino groups in zein are involved incross-linking. Trinitro benzene sulfonic acid (TNBS) was used toestimate the free amino groups in zein before and after cross-linking. Astandard curve was generated with increasing concentration of non-crosslinked and cross-linked zein versus absorbance at 440 nm wavelength.Cross linking efficiency was calculated using the formula,

% of Cross linking efficiency=[a−b/a]×100,

where a=slope of non-cross lined zein versus absorbance, and b=slope ofcross-linked zein versus absorbance. The concentration range of zeinused for constructing the standard curve is 0.357 mg/ml to 12 mg/ml, andcorrelation coefficient is 0.9994.

The extent of cross-linking in zein nanoparticles using differentcross-linking agents is shown in FIG. 18. The cross-linking efficiencyvaried from approximately 70% to approximately 100%. The extent ofcross-linking can be varied by changing the reaction time to range fromapproximately 3 hours to 3 days depending on the cross-linking agent.The cross-linking agent shown here are only examples and the methods ofthe invention are not limited to the use of just the disclosedcross-linking agents. Other cross-linking agents can be used such aspolycarboxylic acids (citric acid or 1,2,3,4-butanetetracarboxylicacid).

Additionally, although the method is illustrated with respect topreparing blank zein nanoparticles, cross-linking may be provided in theformation of nanoparticles containing specific molecules. A specificexample of preparing rhodamine, a water soluble dye, in a nanoparticleis as follows:

Example IX

White zein in an amount of 0.0135 g was dissolved in a mixture of 3 mlof ethanol and 0.25 ml of water (0.25 ml). To the first aqueous solutionwas added 0.005 g of rhodamine-123. The resulting solution was addeddrop-wise into 15 ml of citrate buffer having a pH 7.4 and containing acombination of 0.0675 g of lecithin and (0.135 g) of Pluronic® F68 underconstant application of ultrasonic energy at 1.39 kW/h and 37% amplitudefor 10 minutes with a pulse on-time of 10 seconds and off-time of 1second. During the sonication process, the solution was kept in an icebath to maintain the temperature at about 10° C. Then 0.5 ml ofglutaraldehyde of 25% w/v was added and incubated for 3 hrs at 37° C.while stirring at 300 to 500 rpm. The residual cross-linking agent wasneutralized with 10% w/v sodium metabisulfite. Subsequently, thedispersion was placed on a magnetic stirrer at 300 to 500 rpm at roomtemperature until the ethanol was completely evaporated. After completeevaporation of the alcohol, the nanoparticles were purifiedultracentrifugation. Purification was accomplished by repeated washingwith pH 7.4 citrate buffer and ultracentrifugation using centrifugalfilter of MWt cut off of 5000 Da, at 3950 g for 50 minutes. To theaqueous suspension (pH 7.4 citrate buffer) of rhodamine-loadednanoparticles was added 35 mg of trehalose and the solution was kept at−80° C. to form a solid cake, which was then lyophilized at −47° C. and60 mTorr vacuum for 12 to 14 hrs).

The particle size, polydispersity index and zeta potential of non-crosslinked and cross-linked (using glutaraldehyde as cross-linking agent)rhodamine particles are shown in Table 6.

TABLE 6 Particle Zeta potential Model compound Size (nm) Polydispersityindex (mV) Rhodamine 352 ± 20 0.72 ± 0.20 −14 ± 7 Rhodamine (cross 130 ±35 0.72 ± 0.32 −12 ± 2 linked particles) Each value is a mean of threeexperiments (±SD).

The in-vitro drug release at pH 7.4 is slower when the zeinnanoparticles were cross-linked (FIG. 20) and similarly the enzymaticrelease was also slower (FIG. 21). The cross-linking of the free aminogroups on the surface of zein nanoparticles reduced the particle size,and also reduced the access of solvent and slowed the enzymaticdegradation of the nanoparticles. The cross-linking also significantlyreduced the burst release. Thus cross-linking can further stabilize thenanoparticles and sustain the drug release.

The therapeutic activity and efficacy of the nanoparticles produced bythe method of the invention, can be further enhanced by attachingpolyethylene glycol (PEG) to the nanoparticles. Among the added benefitsof PEGylation is an increase in the circulation half-life of thenanoparticles. An additional advantage of PEG is that it can serve as aspacer to link the targeting ligands, drugs, and imaging agents to zeinnanoparticles, if direct conjugation is not feasible.

FIG. 22 illustrates a method of preparing PEGylated zein nanoparticlesin accordance with another aspect of the method of the invention. Anadvantage of PEGylated zein for making nanoparticles is that it can bemade using only a surfactant, such as Pluronic® F68, as opposed to theuse of a combination of a surfactant and phospholipids for non-PEGylatedzein. A specific method of forming PEGylated zein nanoparticles is asfollows:

Example X

PEGylated zein was produced by adding 0.1 g of methoxy PEG-succinimidylsuccinate (Mwt 5000 Da) to 0.1 g of white zein in 5 ml of 90% ethanol.The mixture was incubated for a period of between three hours and 24hours at 37° C. The solution was then dialyzed (Mwt cut off 10,000 Da)against water in a magnetic stirrer (magnetic stir bar stirred at 100rpm) at room temperature for 24 hours to remove any residual materials.The resulting product was then frozen to −80° C. followed by freezedrying at −47° C. at 60 mTorr vacuum for 12 to 14 hours. The efficiencyof PEGylation observed over various incubation times is shown in Table7, below, where the efficiency percentages were determined using a TNBSassay procedure as described earlier. Other molecular weight PEGs, suchas from 500 to 5000 Da, can be used. Similarly PEG derivatives such asmethoxy PEG-N-hydroxyl succinate ester or other derivatives can be used.

TABLE 7 Incubation time Zein:mPEG ester (hrs) ratio PEGylationEfficiency (%) 24 1:1 65 24 1:2 93 3 1:1 52

Fifty milligrams of PEGylated white zein were dissolved in a mixture of3 ml ethanol and 0.25 ml deionized water. The PEGylated zein solutioncontaining was then added drop-wise into 15 ml of citrate buffer havinga pH 7.4 and containing Pluronic® F68 (0.9% w/v) under constantapplication of ultrasonic energy at 1.39 kW/h and 37% amplitude for 10minutes with a pulse on-time of 10 seconds and off-time of 1 second.During the sonication process the solution was maintained in an ice bathto maintain the temperature at about 10° C. Subsequently, the zeinsuspension was placed on a magnetic stirrer at 300 to 500 rpm at roomtemperature until the ethanol was completely evaporated. Whenevaporation was complete, the nanoparticles were purified. Purificationwas accomplished by repeated washing with pH 7.4 citrate buffer andultracentrifugation using centrifugal filter of MWt cut off of 10000 Da,at 44,000 g for 35 minutes. To the aqueous suspension (pH 7.4 citratebuffer) of zein nanoparticles was added 30 g of 2% w/v trehalose and thesolution was kept at −80° C. to form to solid cake, which was thenlyophilized at −47° C. and 60 mTorr vacuum for 12 to 14 hrs. ThePEGylation process disclosed above may be carried out using highpressure homogenization as disclosed in Example II, above. The sizedistribution of the PEGylated nanoparticles is shown in FIG. 23.

Because zein is a protein, a further advantage of using zein information of nanoparticles is realized in that zein has a large numberof surface functional groups which can be used to attach targetingligands, imaging agents, drugs and other polymers for drug targeting tospecific tissues and other biomedical applications.

Zein nanoparticles formed using the disclosed method may have otheruses, particularly outside of the body. For example, drug-loaded zeinnanoparticles can be used as a coating material for cardiovascular andother biomedical devices. Although described herein with respect to drugdelivery, nanoparticles produced by the disclosed method may be used toencapsulate and sustain the release of molecules of interest to thefood, dairy and cosmetic industries as well. In addition to human drugs,veterinary drugs may also be encapsulated in nanoparticles using thedisclosed methods. Zein nanoparticles may be used to protect moleculesfrom adverse environmental agents such as moisture, oxidation, lightetc. This utilization may include molecules of interest to thepharmaceutical, food, dairy and cosmetic industries.

Zein can be combined with other natural and synthetic polymers to designnovel nanoparticles with unique properties for various applications inthe biomedical, pharmaceutical, food, dairy and cosmetic industry. Forexample, by attaching a pH-sensitive polymer or linker to zein, the zeinnanoparticles can be made to release the drug in response to a pHstimulus.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to thesame extent as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein, including:

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1. A method of producing non-immunogenic nanoparticles, comprising:providing a hydrophobic water-insoluble protein; dissolving said proteinwith a hydroalcoholic solvent to provide a first aqueous phase solution:adding a buffering agent to the first aqueous phase solution in thepresence of a surfactant and a phospholipid to produce a second aqueousphase solution having a pH of between approximately pH 6.8 andapproximately pH 7.4; processing said second aqueous phase solution toeffect a reduction in diameter size of particles within the solution;evaporating any residual solvent to produce nanoparticles having adiameter size of less than approximately 400 nm; and isolating saidnanoparticles.
 2. The method according to claim 1 further comprisinglyophilizing the nanoparticles following isolation.
 3. The methodaccording to claim 2 further comprising storing the nanoparticles underconditions that restrict exposure of the nanoparticles to atmosphericpressure.
 4. The method according to claim 1 wherein said base proteinis a selected grade of zein.
 5. The method according to claim 4 whereinsaid selected grade of zein is white zein.
 6. The method according toclaim 1 wherein said buffering agent is citrate buffer.
 7. The methodaccording to claim 6 wherein said surfactant is a poloxamer and saidphospholipid is lecithin.
 8. The method according to claim 7 wherein theratio of said surfactant to said phospholipid is about 2:1.
 9. Themethod according to claim 1 wherein said processing of said secondaqueous phase solution to effect a reduction in diameter size ofparticles further comprises subjecting the nanoparticles to ultrasonicshear, high pressure homogenization or a combination thereof.
 10. Themethod according to claim 1 further comprising adding, to said proteinin the formation of the first phase solution a molecule that is selectedfor nanoparticle encapsulation.
 11. The method according to claim 10wherein said molecule is a therapeutic substance selected foradministration to a subject.
 12. The method according to claim 11further comprising administration of said nanoparticle to a patient. 13.The method according to claim 10 wherein said protein is PEGylated. 14.(canceled)
 15. The method according to claim 1 further comprisingcross-linking said nanoparticles.
 16. (canceled)
 17. (canceled)
 18. Amethod of administering a therapeutic molecule to a subject comprising:administering to a subject suffering from a disease or condition thetherapeutic composition of claim 16, thereby treating the disease orcondition.
 19. (canceled)
 20. The method according to claim 12 whereinsaid administrating of said nanoparticle is by oral, parenteral,intravenous, intraperitoneal, subcutaneous, topical, nasal oropthalmological routes of administration.
 21. (canceled)
 22. (canceled)